1. Field of the Invention
This invention relates to monoclonal antibodies derived from crosslinked fibrin derivatives and assays for said cross linked derivatives which may be used as a diagnostic test for fibrin breakdown products in fibrinolysis in general and pre-thrombotic and thrombotic states including Disseminated Intravascular Coagulation (DIC).
2. Description of the Related Art
Fibrinogen is a large protein molecule that normally circulates in the blood plasm in the dissolved state. Under attack from the enzyme thrombin, the fibrinogen molecules link up, spontaneously aligning themselves into a long thread like polymer or network called fibrin which is the primary ingredient of blood clots.
It has been discovered that upon digestion with an enzyme called plasmin (which functions in the blood to destroy the fibrin network and restore the fluidity of the plasma), fibrinogen breaks down into fragments designated by A-E. Fragments D and E made up the bulk of the recovered mass and there was about twice as much D as there was of E. Fibrogen also has been discovered to have a trinodular shape wherein E is a central component and D is a terminal component.
Plasmin digests of fibrin and fibrinogen can be differentiated from each other using polyacrylamide gel electrophoresis (PAGE). Crosslinking of fibrin with an enzyme called Factor XIIIa forms dimers of fragment D called D dimer. Factor XIIIa is an enzyme which introduces covalent bonds between adjacent monomers in fibrin and thus may stabilize the fibrin structure. For a more detailed explanation of the nature of the crosslinking between fibrin monomers, refer to Budzynski et al, Blood, Vol 54, No. 4 (October)1979. Factor XIIIa is activated by the thrombin-catalyzed removal of a peptide from a precursor in the plasma and in blood platelets. D dimer is a molecule of about 189,000 daltons which consists essentially of two fragment D moieties derived from different fibrin molecules covalently bound by cross link bonds between the gamma chain remnants of fibrinogen. Fibrinogen itself comprises 6 chains including two copies of an alpha, beta and gamma chain.
Another complex (DD)E is formed by plasmin degradation of cross linked human fibrin and comprises a combination of two D fragments and fragment E.
Other cross linked derivatives may be prepared as described in an article from Seminars in Thrombosis and Hemostasis Vol 8, No. 1 (1982) entitled "Detection and Relevance of Crosslinked Fibrin Derivatives in Blood" by Graeff and Halfer. These include high molecular weight cross linked derivatives and may be referred to in the above reference as derivatives DY, YY, XD, XY, DXD and YXD.
Normal hemostosis or coagulation of the blood involves maintaining intravascular constituents in a liquid phase or suspension while concomitantly permitting local deposition of solid phase blood components in areas of vessel damage. In a healthy individual, it has been assumed, but never experimentally demonstrated, that a balance exists between a low-grade intravascular deposition of fibrin and its removal by fibrinolysis or cellular phagocytosis.
Early clinical observations revealed that some severly ill patients developed signs of hemorrhage and massive bruising and had prolonged clotting times and thrombocytopenia. After death, in some cases, fibrin thrombi were demonstrated in the microvasculature. The diffuse nature of these thrombi gave rise to the term "disseminated intravascular coagulation" (DIC). Subsequently, coagulation factors were shown to be reduced. These findings give rise to the concept of "consumptive coagulapathy", a term sometines used as a synonym for DIC.
The currently accepted sequence of events in DIC involves activation of the coagulation system resulting in platelet consumption, thrombin generation, fibrin deposition, and secondary fibrinolysis. The net biologic effect of this process reflects a balance between fibrin deposition and fibrin clearance. The resulting clinical manifestations may be hemorrhage, when depletion of coagulation factors predominates, or ischemic tissue damage due to the effects of vascular occlusion.
DIC has been reported as a secondary phenomenon in a wide variety of disorders, particularly those accompanied by a combination of shock, acidosis, and hypoxemia. The well-recognized clinical associations are sepsis, major trauma, malignancy, and obstetric disorders. In these clinical settings activation of the coagulation sequence results in consumption of coagulation protein and platelets, leading to fibrin deposition in the micro-circulation. The precise factors that initiate the DIC are unknown, but many potential mechanisms have been demonstrated in animal experiments.
Ideally a definitive diagnosis of DIC should require the direct demonstration of diffuse fibrin deposition. The practical difficulty of obtaining multiple direct biopsy evidence to differentiate between localized and generalized fibrin formation has led to the development of indirect tests that are substituted as diagnostic end points. However these tests are not specific for the syndrome of intravascular fibrin deposition. There specificity is further reduced by the action of other enzymes that although are not able to convert fibrinogen to fibrin can cause similar alterations to thrombin on the other coagulation factors involved in thrombosis. All of the direct tests are based on the principle that thrombin is the only enzyme (snake venoms excluded) capable of converting fibrinogen to fibrin in man.
Also apart from the paracoagulation tests that detect the presence of circulating soluble fibrin monomer complexes, none of the more specific thrombin specific tests is readily available or useful for immediate clinical application in the diagnosis of clinical DIC. These tests include the FPA (fibrinopeptide A) test where FPA is measured by a specific RIA procedure, fibrin monomer assays, fibrinogen gel exclusion chromatography and tests for FPB (fibrinopeptide B) or thrombin increasable FPB.
Tests with biochemical nonspecificity for thrombin action include the prothombin time (PT) thromboplastin time (A PTT) and thrombin clotting time (TCT) tests. Although frequently useful in practice it must be recognized that information obtained from these tests is nonspecific in nature, acting as a measure of clotting factor depletion regardless of etiology.
Coagulation factor assays have also been found to be relatively non specific and these include assays for cofactors V and VIII as well as tests for fibrinogen levels.
Tests for fibrin-fibrinogen degradation products so far have not proved to be specific for the action of plasmin on fibrin and may yield positive results where there has been fibrinogenolysis without prior thrombin action on the fibrinogen molecule. These tests include tests for fragments D and E.
Tests for thrombin-mediated platelet interaction or release have been found to be nonspecific in nature. These include platelet count, platelet survival and tests of platelet release.
The use of radio labelled fibrinogen in relation to identifying clotting factors have also been attempted but found to be time consuming and difficult to perform.
Thus, in summary of the prior art, the efficacy of a diagnostic test lies in the ability to indicate the presence or absence of disease. There are well recognized essential design principles for studies determining the efficacy of a diagnostic test which enables the four indices of sensitivity, specificity, positive predictive value, and negative predictive value to be determined. The first requirement is the adoption of a suitable standard for diagnosis. Ideally, this standard should be slightly more than a clinical definition and should be as specific as possible for the disease entity. An inherent difficulty in relation to DIC is the absence of a comprehensive definition of this disorder. The clinical picture is very nonspecific. Many of the routinely available laboratory tests also lack diagnostic specificity. A low platelet count suppots the likelihood of DIC but may occur as an isolated finding secondary to infection. Similar limitations apply to many of the coagulation assays. Hypofibrinogenemia does not distinguish between primary fibrinolysis, due either to the action of plasmin or elastases, and secondary fibrinolysis following the thrombin-medicated conversion of fibrinogen to fibrin. Alternatively, sensitive tests of thrombin action are available, but there are obvious drawbacks with their clinical use. An example is the FPA assay, which, although specific for thrombin action, is exquisitely sensitive and may detect localized intravascular coagulation yielding a positive result in uncomplicated venous thrombosis. The clinical significance of an elevated FPA level, even with a positive paracoagulation test, is then at issue, particularly if the platelet count, global clotting tests, and fibrinogen level are normal.
For these reasons, sensitivity, specificity, and predictive values cannot be determined in a standard fashion. The clinical presentation of the disorder is complex and unpredictable. The application of the available tests for diagnosis are therefore best considered in relation to the different clinical syndromes of intravascular coagulation.
It has also been proposed to assay for D dimer as a diagnostic test for DIC. However, this has necessitated the use of PAGE as described previously and this technique is far too cumbersome for routine clinical use. Antibodies have been raised to fibrin derived D-D-E fragments but in their current form these cross react with fibrinogen fragment D derivatives and as yet are unsuitable for clinical use.
A useful summary of DIC and conventional diagnostic tests will be found in Seminars in Thrombosis and Hemostasis Vol 8, No. 3(1982) and an article entitled DIC; The Application and Utility of Diagnostic Tests by Ockelford and Carter.
In the abovementioned Budzynski reference describes the study of polyclonal anti-D dimer antibodies using two different antisera. In this test antibodies were raised against specific markers on the D dimer molecule. In the test antisera were obtained in chickens and rabbits against a mixture (1:1) of D.sub.2 E complex and D dimer and against D dimer exposed to 3M area at pH 5.5. It was however stated in this reference that it was hoped by the authors that the results of this test could be applied to clinical situations, such as the distinction between disseminated intravascular coagulation and primary fibrinogenolysis, since circulating fragment D dimer should be present in the former conditions but not in the latter. However, such application would require a much higher difference in reactivity between fragments D dimer and D, since this and other relevant clinical states have high concentrations of circulating fibrinogen derivatives in addition to the crosslinked fibrin fragments. It was also considered that although it was shown that the assays could be performed even in the presence of enormous concentrations of fibrinogen, further development of the antibody specificity was needed before it can be reliably applied to the relevant clinical situations.
The abovementioned Graeff and Hafter article also points out that crosslinked fibrin derivatives in blood such as D dimer may be considered as a marker for DIC. However, there is nothing in this article to demonstrate that a reliable diagnostic test for DIC could be devised based on crosslinked fibrin derivatives.
It is therefore an object of the present invention to provide an assay procedure for crosslinked fibrin derivatives which may be used on a clinical basis.